Mosquito-borne members of the family Bunyaviridae are recognized as a major cause of pediatric encephalitis in North America with 70-130 symptomatic cases each year. Clinically important viruses include La Crosse virus (LACV) and Jamestown Canyon (JCV) in the US, and Tahyna virus (TAHV) in Europe. The majority of LACV infections are mild and never reported, however, serologic studies estimate infection rates of 10-30/100,000 in endemic areas. LACV encephalitis has become the most commonly reported pediatric arboviral encephalitis in the US with 70-130 symptomatic cases a year with severe sequelae. For these reasons we are interested in developing a vaccine to prevent this CNS disease. LACV sequence analysis: To better understand the genetic factors contributing to disease, the full genome sequence of the five known human isolates of LACV and of 13 mosquito isolates was analyzed, and a high level of genetic relatedness amongst the 18 viruses was observed. Two major LACV genetic groupings were identified that had allelic differences in the S, M and L genome segments. Group 1 viruses were underrepresented in mosquitoes (1 of 13 isolates) but predominant in humans (4 of 5 isolates), while Group 2 viruses were isolated predominantly from mosquitoes. The presence of Group 1 viruses in 80% of humans with severe central nervous system (CNS) disease suggested that Group 1 viruses possess a higher encephalitogenic potential than Group 2 viruses. To explore this hypothesis, viruses from both genetic groups were analyzed for their neurovirulent phenotype in mice, and a representative virus from each genetic group was analyzed in nonhuman primates. Whereas the viruses from both genetic groups exhibited somewhat similar neuroinvasiveness and neurovirulence in mice, the primate model demonstrated that Group 1 human virus possesses higher neurovirulence, as defined by greater levels of virus replication in the CNS and more severe virus-induced neuroinflammation. This study confirms the observation that Group 1 LACV is more likely to be associated with CNS disease. Therefore, the low incidence of encephalitis due to LACV infection in humans may be partially explained by the observed low frequency of neurovirulent Group 1 LACV among mosquitoes. Conversely, the greater prevalence of less pathogenic Group 2 LACV in mosquito populations may account for the asymptomatic or mild nature of the majority of LACV infections in humans. A detailed study of the neuropathogenesis of LACV for the CNS of non-human primates is ongoing. JCV sequence analysis: We have completed the sequence of three isolates of JCV collected in geographically diverse locations over a 57 year time span. The nucleotide identity for the three JCV viruses is 90, 83, and 85% for the S, M, and L segments, respectively ,whereas the percent identify for the predicted amino acid sequences of the N, NSS, M poly, GN, NSM, GC, and L proteins is 97, 91, 94, 98, 91, 94, and 97%, respectively. In Swiss Webster mice, each JCV isolate exhibits low neuroinvasiveness but high infectivity. Two of the three JCV isolates were highly neurovirulent after IC inoculation whereas one isolate, JCV/03/CT, exhibited low neurovirulence. In rhesus monkeys, JCV infection is accompanied by a lack of clinical disease, a low-titered viremia, and a robust neutralizing antibody response. A relatively high level of amino acid sequence conservation was observed even for viruses isolated 57 years apart indicating that the virus is in relative evolutionary stasis. JCV is highly infectious for mice and monkeys, and these animals, especially mice, represent useful experimental hosts for further study. TAHV sequence analysis: In order to determine the genomic sequence of wild-type TAHV, we chose three geographically distinct TAHV isolates isolated over a 26-year period and are the first to determine the complete sequence of the TAHV S, M, and L segments. TAHV maintains a highly conserved genome with both nucleotide and amino acid sequence identity greater than 99%. In order to determine the extent of genetic relatedness to other members of the CEV serogroup, we compared protein sequences of TAHV with LACV, Snowshoe Hare virus (SSHV), Jamestown Canyon virus (JCV), and Inkoo virus (INKV). By amino acid comparison, TAHV was most similar to SSHV followed by LACV, JCV, and INKV. The sequence of the GN protein is most conserved followed by L, N, GC, NSS, and NSM. In a weanling Swiss Webster mouse model, all three TAHV isolates were uniformly neurovirulent, but only one virus was neuroinvasive. In rhesus monkeys, the virus was highly immunogenic even in the absence of viremia. Cross neutralization studies utilizing monkey immune serum demonstrated that TAHV is antigenically distinct from North American viruses LACV and JCV. However, we have also demonstrated significant cross-neutralization which may complicate virus identification since anti-JCV serum neutralized both LACV and TAHV. These data also suggest that a single vaccine could generate a cross-neutralizing antibody response which may provide protection against CEV serogroup viruses from a wide geographic range. Vaccine development: Given our experience with antigenic chimeric viruses of the various flaviviruses, we sought to explore the possibility that we could use an attenuated LACV as a backbone to create antigenic chimeric viruses in which the coding region of the M segment of LACV is replaced with that of a second bunyavirus. In our efforts to create live-attenuated viral vaccine candidates for this virus group, we have generated a recombinant La Crosse virus expressing the attachment glycoproteins of Jamestown Canyon virus. The JCV/LACV chimeric virus contains full-length S and L segments derived from LACV. In the M segment, the ORF of LACV is replaced with that derived from JCV and is flanked by the non-coding regions of LACV. Chimerization did not affect the growth of the chimeric virus in tissue culture, and the virus remains highly infectious and immunogenic in Swiss Webster mice. Although both LACV and JCV parental viruses are highly neurovirulent in 21 day-old mice, with LD50 values (log10 PFU) of 0.1 and 0.5 respectively, chimeric JCV/LACV is highly attenuated and does not cause disease even after intracerebral inoculation of 3 log10 PFU. Parenteral vaccination of mice with 3 log10 PFU of JCV/LACV protected against lethal challenge with LACV, JCV, and Tahyna virus (TAHV). The chimeric virus was infectious and immunogenic in rhesus monkeys and protected against the development of viremia after JCV challenge. The use of the LACV genetic background to deliver M segment protective antigens from pathogenic Bunyaviruses other than JCV could be an efficient method for rapid development of vaccines effective against pathogenic bunyaviruses, including Rift Valley fever virus and Hantavirus.